In vitro efficacy of essential oils against Sarcoptes scabiei

The mite Sarcoptes scabiei is responsible for scabies, a pruritic and contagious skin disease in humans. S. scabiei is also responsible for mange in a wide range of animal species. The treatment of S. scabiei infection is hampered by an under-effectiveness of the few available drugs. The objective of this work was to evaluate the in vitro acaricide activity of a large number of plant essential oils (EOs) against S. scabiei. EOs were selected mainly on the basis of traditional treatments for dermatological infections in Madagascar. The sarcoptes originating from a porcine animal model were tested at concentrations ranging from 10 to 0.1%. The viability of sarcoptes was assessed by stereomicroscopic observation at 5 min, 15 min, 30 min, 45 min and then every hour until 6 h after treatment. Estimates of lethal time and lethal concentration producing 50% mortality were generated using a probit analysis. The survival curves were estimated using the Kaplan Meier method. A total of 31 EOs from different plants were tested. Cinnamomum zeylanicum (cinnamom) and Ocimum sanctum (tulsi) oils were the most active for all concentrations tested. They may be included in in vivo studies, in order to further assess their potential interest as topical treatments.


Materials and methods
Essential oils. Essential oils of these plants were selected on the basis of historical reports on traditional treatments of dermatological infections in Indian Ocean area. Other EOs were chosen according to bibliographical data [22][23][24][25][26] . EOs were provided by the Centre National d' Application de Recherche Pharmaceutique, Tananarive, Madagascar, or were purchased for those commercially available ( Table 1). EOs of two plants, Psiadia altissima and Tetradenia nervosa, extracted at CNARP Madagascar, are not wild or endemic plants and are not species at risk of extinction. The use of these plant species has complied with relevant institutional, national, and international guidelines and legislation.
EOs were prepared for assays by diluting them in paraffin oil which was also used as control. The dilutions were performed right before setting for contact or fumigation bioassays.
Collection of mites. S. scabiei mites were collected from infected pigs maintained at the Centre de Recherche Bio-Médicale, Maisons-Alfort, France. Pigs were experimentally infested as previously described 27 . All animals were maintained in strict accordance with good animal practices as defined by the French and European code of practice for the care and use of animals for scientific purposes (approval No. 02515.01). The study protocol was approved by the « Comité d' éthique en expérimentation animale Anses/EnvA/UPEC » (the local ethics committee for animal experiments). The study was carried out in compliance with the ARRIVE guide- www.nature.com/scientificreports/ lines. Mites collected from the ears and back of pigs were placed in Petri dishes. All Petri dishes were incubated at 35 °C at least 30 min before experiments. Mites were observed under a stereomicroscope (Nikon©, SMZ645, Lisses, France) and motile stages (adults and larvae) were included in the assays.

Contact bioassays.
The acaricide activity of the EOs was evaluated by bioassays as described by Fang et al.
with slight modifications 28 . In a first screening, all EOs were diluted with paraffin oil at 10% and 5%. The evaluation was conducted further for EOs showing acaricidal activity in contact bioassays in less than 3 h at the 5% concentration. These EOs were then diluted with paraffin oil at 1%, 0.5% and 0.25% concentrations. For each experiment, adult and larvae stages (n = 10) were collected with a needle and placed directly in plastic Petri dish (5 cm in diameter) in direct contact with 50 µl of each diluted solution. All experiments were done in triplicate. Negative control mites were treated with paraffin oil and clove essential oil was used as positive control as previously described 28 . All dishes were incubated at room temperature and observed under a stereomicroscope at 5 min, 15 min, 30 min, 45 min then every hour to 6 h post-treatment. The mites were regularly stimulated with a needle to determine their viability. When they did not respond to stimulation, they were recorded as dead.
Fumigation bioassays. The vapor phase toxicity of all EOs was investigated. For each fumigation bioassay, 10 mites of motile mixed stages were placed at the bottom of a plastic Petri dish (5 cm in diameter). A covering filter paper (Whatman, No. 2,5 cm in diameter) was put on the lid of the Petri dish and treated with 200 µl of EOs diluted in paraffine oil at concentration of 5% and 1%. Negative control received paraffin oil alone and tea tree oil was used as a positive control as described by Fang et al. 28 . Three replicates were performed for each concentration. Mortality was recorded after 3 h. The mites were stimulated with a needle to determine their viability. When they did not respond to stimulation, they were recorded as dead.
Ovicidal activity. Cinnamomum zeylanicum and Ocimum sanctum 5% oils were selected for evaluating their ovicidal activity. Scabies eggs were counted into groups of 10 and were placed directly in the bottom of Petri dish (5 cm in diameter) with 50 µl of each solution or paraffin oil for control. The dishes were incubated at 30 °C under 75% relative humidity for 5 days. The number of hatched and unhatched eggs were counted under microscope at the end of the 5-d period.
Gas chromatography/Mass spectrometry analysis. The main components of the EOs were analyzed by gas chromatography/mass spectrometry with an Agilent 5977A Series GC/MSD System as previously described 28 .
Statistical analyses. Probit regression estimates and lethal time and concentration producing 50% mortality in all stages were generated using probit analysis 29 . Survival curves were estimated using the Kaplan Meier method. The 95% CI were calculated using a normal distribution. Difference in the survival curves were assessed using the Log-rank test adjusted with Tukey's method. The median lethal time (LT 50 values) of EOs was calculated using a generalized linear model with binomially distributed error term. R v 3.5.1 and SAS v9.4 were used for all analyses. The significant level was set at p < 0.05. Ethical approval. Mites were collected from experimentally infected pigs which were maintained in strict respect to the guidelines as defined by the French and European code of good practice for the care and use of animals for scientific purposes (French Ministry of Research approval no: 0251503).

Results
The principal components of EOs used in this study are shown in Fig. 1.
There was no mortality of S. scabiei in paraffin oil. The survival curves for the different concentrations are shown in Fig. 2.
In pre-screening of all EOs, in contact bioassays, only four EOs (katrafay, turmeric, turpentine and lantana) showed no acaricidal property at 10% concentration (Table 2).
At 5% concentration, 14 out 31 EOs showed acaricidal property. Five EOs were still effective at 1% concentration. Two Eos (tulsi and cinnamom) remained acaricidal at 0.5% concentration (and even at 0.25% concentration for tulsi oil). The LT 50 values of EOs tested at 1%, 0.5% and 0.25% concentrations are shown in Table 3.
All mites were killed within one hour with tulsi oil and cinnamom oil diluted at 1%. The cinnamom and tulsi oils exhibited also the highest percentage of mortality at 0.25% concentration over an exposure period of 6 h. Tulsi oil and cinnamom oil were the most active oils for all concentrations tested, followed by lemon myrtle, litsea, bourbon geranium, rosewood and ahibero.
The results of the fumigation assay were not completely in accordance with those of the fumigation assay, though most effective EOs in contact assays were also the most effective in fumigation assays (Table 2). Among EOs which were not acaricide at 5% concentration in contact assays, only combawa showed efficacy by fumigation at 5% concentration. Interestingly, tulsi and cinnamom, showed equal efficacy by contact and fumigation. Nevertheless, these two EOs demonstrated no ovicidal activity in comparison with the negative control group (Data not shown).

Eucalyptus globulus
Eucalyptus globulus VA18 www.nature.com/scientificreports/ large biodiversity, either animal or vegetal, with the presence of many endemic species. Traditional medicine is also well developed in these areas. Scabies can manifest with a wide spectrum of clinical signs and variable severities, making clinical diagnosis difficult even for a medical staff. The condition is not fully recognized or characterized among indigenous populations. Thus, in our search for potentially interesting plants in the literature, we selected a large acceptation of terms such as "pruritus" or "dermatological affection".
Results obtained in vitro with tested EOs are not necessarily reproducible from one study to another. EOs may vary in the nature and the concentration of their compounds within a certain range. These variations can be attributed to factors that influence the plant's environment, such as climate, geographical location, soil type and fertilizer used 30,31 and also to the chosen method of extraction. Thus, it is important to analyze the composition of the EOs tested in order to characterize the efficacy of most active EOs. This approach permits to identify the major components which are probably responsible for most of the effects of the EOs. Only two EOs of our series have been previously tested: one obtained from Pelargonium asperum 28 and another one from Citrus limonum 32 . While the EO from Pelargonium asperum showed one of the best efficacies of our series, that from Citrus limonum was one of the less effective. These results were consistent with those of the previous studies 28,32 . Not surprisingly, citronellol was the major component of the EO from Pelargonium asperum found in both our study and that of Fang et al. Similarly, limonene was the main component found in the EO from Citrus limonum of our series and of that of other studies.
The biological properties of essential oils are usually determined by their major components 15,28,32 . Various major components were found in the present study according to the EOs tested. In particular, geranial and neral were the main components of lemon myrtle and litsea which were among the most effective EOs tested. Geranial and neral were also the main components of the lemongrass (Cymbopogon citratus) oil which has been reported as a promising agent for scabies control 21 . Likewise, eugenol was found as the main component of tulsi and cinnamom EOs, the most effective EOs of the present series. Eugenol was also the main component of clove oil which was found as the most scabicide agent in a previous, though more limited, series 28 . The present results confirmed the potential interest of geranial, neral and eugenol as scabicide agents. Actually, a drug containing eugenol (at a 2.5% concentration) is already on the Chinese market for scabies treatment since 1990s 33 .
Cinnamom oil has never been tested on S. scabiei. Its efficacy against Psoroptes mites, major parasites of ruminants or rabbits, has been demonstrated in vitro and in vivo studies 34 . The sample of Cinnamom oil used in the present study contained, besides eugenol, its majoritory compound, a high concentration of benzyl benzoate. Thus, it is not surprising that it appeared among the most effective EOs tested.
As previously reported 28 the fumigant effect of EOs did not correspond exactly to their efficacy by direct contact. In the present study, 1,8-cineole, contained in several EOs, appeared more active by fumigation than by contact. This has been already shown as 1,8-cineole was the main component of eucalyptus oil 28 . Linalol, a major component of rosalina, coriander and camphorwood, and citronellal, a major component of combawa and eucalyptus citriodora, appeared also more active by fumigation than by contact in the present study. To our knowledge, this was not previously described.
We chose to study the ovicidal efficacy only for the most active EOs of our series on the basis of contact and fumigation assays because a compound having only ovicidal efficacy would probably be of limited therapeutic potential. Unfortunately, tulsi and cinnamom EOs showed no significant ovicidal efficacy. It may be underlined that current first line scabicides have no ovicidal activity either. Accordingly, potential formulations including these EOs with a therapeutic purpose would require two applications separated by a 10-14-day interval as it is the case for most current treatments.
We chose to use paraffin oil (mineral oil) to perform the dilutions of EOs and as treatment for negative control groups. Paraffin oil was the solvent the most frequently used as diluent for evaluating the effects of EOs against Sarcoptes mites in previous studies 20,21,28,35,36 . It has been demonstrated that paraffin oil has no toxicity against mites.
EOs are generally well tolerated as demonstrated by their millenary use in aromatherapy 37 . They are reported as slightly toxic' or 'non-toxic' , with an oral and dermal LD 50 (Median lethal dose) value of 1-5 g/kg or more than 5 g/kg to mammals 38 . Understandably, the toxicity depends on the nature of the components and on their concentrations 38 . The EOs showing best scabicide efficacy by contact and fumigation assays, tulsi and cinnamom, would probably be well tolerated at concentrations ≤ 1% for topical use, though an allergic contact dermatitis may occur independently of the concentration.
Scabies affects often young children, particularly children aged 1-24 months 39 . However, first line treatments, permethrin and ivermectin, are not recommended for use in young children 40 . Some EOs within a certain range of dilution rates could be used in young children. Some dilution rates of EOs have been proposed according to age groups : 0.10% to 0.20% in full-term infant, aged 0-3 months ; 0.25-0.50% for 3-24 month-old children ; and 1-2% for 2-6 year-old children 38 . Tulsi and cinnamom EOs appeared effective in vitro within these ranges of dilution.
Owing to their multiple active components, EOs are less likely to lead to resistance comparing to conventional drugs 19 . In addition to their scabicide property, EOs may have various biological properties including antibacterial, anti-inflammatory, and antipruritic effects 16 . All these adjuvant properties are particularly interesting for the treatment of scabies.
In conclusion, there has been a growing body of evidence indicating the potential value of EOs as control agents against a range of ectoparasites, including S. scabiei. The present study demonstrated the contact and fumigant effects of new EOs which may be useful to incorporate in novel topical formulations for scabies control. Our results confirmed the scabicide efficacy by contact of some components as eugenol, geranial and neral. Other components as linalol and 1,8-cineole appeared more effective by fumigation. Two EOs, tulsi and cinnamom, proved to be more acaricide than others, though they appeared to be devoid of ovicidal activity. These two EOs